CN110680776A - 侧柏的应用 - Google Patents
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- CN110680776A CN110680776A CN201810731150.8A CN201810731150A CN110680776A CN 110680776 A CN110680776 A CN 110680776A CN 201810731150 A CN201810731150 A CN 201810731150A CN 110680776 A CN110680776 A CN 110680776A
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Abstract
本发明涉及植物提取物技术领域，公开了侧柏的应用。本发明发现中天然植物侧柏提取物能够显著地上调有助于皮肤细胞、组织修复的基因sirt1、nidi1和igf1，同时能够显著增加表皮的厚度，使基底层上细胞排列更加紧密，角质层分化更加完善，表明侧柏提取物可以提升皮肤整体活力和健康状况，具有修复皮肤组织的能力，进而可以不断的促进皮肤细胞增殖和皮肤组织的再生，延缓各种现有皮肤细胞活力下降、数量减少导致的衰亡现象。
Description
技术领域
本发明涉及植物提取物技术领域，特别涉及侧柏的应用。
背景技术
皮肤被覆盖于整个人体表面，与外界环境直接接触，除了保护机体免受外界各种因素的伤害外，又是反应全身健康状态的一面镜子。人的皮肤由一些区组成，这些区中的三个覆盖了整个人体，即浅层区(浅层区为表皮)、真皮、以及深层区(深区为皮下组织)。表皮是一种角质化的分层的扁平上皮组织。它主要由角化细胞和其它细胞组成且黏附在基底膜上，基底膜将表皮与真皮隔开。真皮是一种结缔组织。它的结构源自细胞外基质的成分和成纤维细胞之间的排列和交互作用，该成纤维细胞进行细胞外基质的成分合成和降解。真皮构成皮肤的主要部分。真皮分为两层，乳头层和网状层。真皮由胶原纤维、弹性蛋白纤维、糖胺聚糖和蛋白聚糖组成。这些不同的结构形成了在对皮肤的生物力学性能起到关键作用的复杂网络。最后，下皮，为最深层区且一般也是皮肤的最厚区，内陷到真皮上，且通过胶原蛋白和弹性蛋白纤维附着至上面覆盖的真皮上。下皮实质上由专门积累和储存脂肪的细胞，即脂肪细胞构成。在外皮覆盖层未被角质化的区域中，表皮也被称为粘膜。上皮也包括在真皮基质中的成纤维细胞。
皮肤衰老是人体正常的生理现象，明显特征表现为出现皱纹，身体暴露部位皮肤变得粗糙、皱纹加深加粗，色素沉着、出现老年斑，肌肤弹性不再、变得松弛等。这主要是由于随着年龄的增长，皮肤的真皮层中成纤维细胞数量逐渐减少、合成胶原蛋白和弹性蛋白的能力下降，表皮细胞更新减慢、屏障功能减弱，角质形成细胞活力下降，继而导致了皮肤干燥、脱屑、皱纹等。
目前关于皮肤衰老机制的研究中，代表性的解释是自由基衰老学说和氧化应激理论，即机体中自由基过量的原因导致了皮肤的衰老。机体在遭受各种有害刺激时，氧化速率超出氧化物的清除速率，氧化系统和抗氧化系统失衡，也就是机体内的自由基代谢处于不平衡状态时，过量的自由基引起机体损伤，破坏细胞膜及其他重要成分，引起膜脂过氧化，导致蛋白质损伤，细胞基质合成减少，皮肤失去弹性、产生皱纹。为了解决这个问题，目前通常的做法是使用自由基清除成分来保持机体内氧化系统和抗氧化系统的平衡，其可以一定程度上延缓肌肤衰老的问题。
但这些自由基清除成分通常吸收效果较差，限制了其抗衰老的功效。故而市面上出现了一些试图改善这个问题的途径，比如抗氧化成分的简单叠加组合或者外用高分子成分来暂时收紧皮肤，使得更多的自由基清除成分能被锁在皮肤中。但是由于有的成分难以吸收，无法发挥其作用，简单的叠加也无法起到成倍的效果，甚至可能抵消；而外用的大分子物质在水洗后就没有了效果。这两种方法均不能有效地解决上述的自由基清除成分吸收较差的问题。
发明内容
有鉴于此，本发明的目的在于提供侧柏在制备皮肤组织修复产品中应用，更具体地是提供侧柏提取物在制备皮肤组织修复产品中的应用；
本发明的另外一个发明目的在于提供侧柏在制备延缓皮肤衰老产品中应用，更具体地是提供侧柏提取物在制备延缓皮肤衰老产品中的应用；
侧柏(学名：Platycladus orientalis(L.)Franco)属常绿乔木。木材可供建筑和家具等用材，叶和枝入药，可收敛止血、利尿健胃、解毒散瘀；种子有安神、滋补强壮之效。目前，在皮肤组织修复方面在国内外至今尚未报道。
沉默信息调节因子2相关酶1(sirtuin1，SIRT1)是著名的长寿基因之一。Sirt1是依赖于尼克酰胺腺嘌呤二核苷酸的组蛋白和非组蛋白去乙酰化酶，为Sirtuins家族成员之一。它与细胞分化、衰老、凋亡和能量代谢密切相关，参与机体长寿、能量代谢、应激、炎症以及肿瘤的调控等。在皮肤中的研究发现，Sirt1具有提高皮肤修护能力的作用。比如Sirt1可保护皮下胶原蛋白，防止紫外线照射引发的光老化；它还可下调STAT3、环氧化酶2等细胞因子，发挥抗炎作用；此外Sirt1还具有抑皮肤癌的功能。Sirt1的激活剂可能在今后皮肤病的治疗和预防中发挥重要的作用。一些国外的化妆品公司将对Sirt1调节作为重要靶标用于研发提高皮肤修护能力。
巢蛋白nidi1(Nidogen1)基因编码的蛋白质为巢蛋白，巢蛋白是DEJ的重要蛋白，起着连接Ⅳ型胶原和层粘连蛋白5的作用，对DEJ的结构和功能是必不可少的。而DEJ结构和功能的正常与否，决定着皮肤能否正常更新和分化。巢蛋白nidi1(Nidogen1)基因编码基底膜糖蛋白的巢蛋白家族成员。蛋白质与基膜的几种其他成分相互作用，并且可能在与细胞外基质的细胞相互作用中起作用。因此，巢蛋白nidi1基因的表达与皮肤组织修复息息相关。
胰岛素样生长因子igf1(insulin like growth factor 1)由该基因编码胰岛素样生长因子1，该蛋白质在功能和结构上与胰岛素相似，并且是参与调节生长和发育的蛋白质家族的成员。胰岛素样生长因子1具有促进角质细胞迁移，促进创面再上皮化，影响皮肤细胞的生长和发育，胰岛素样生长因子1水平的上调有助于皮肤组织修复。
本发明通过人原代成纤维细胞(NHDF)及人原代角质细胞(NHEK)构建可用于检测的3D皮肤模型，在皮肤模型培养至3天按照测试浓度加入侧柏提取物溶液，并以未加提取物溶液的模型为对照，18天后提取测试组和对照组皮肤模型中RNA进行检测，结果显示，添加侧柏提取物(浓度为10μg/mL)后，与对照组(NT)相比，在皮肤模型中基底层相关蛋白nid1基因表达上调1.74倍，igf1基因表达上调1.74倍，sirt1基因表达上调1.53倍；添加侧柏提取物(浓度为100μg/mL)后，与对照组(NT)相比，与基因nid1表达上调1.35倍，两个浓度下的侧柏提取物可以诱导皮肤组织修复相关基因的表达，可提高皮肤细胞整体修复能力。
同时，3D皮肤模型切片快蓝染色试验结果显示，在添加侧柏提取物后，对比未处理组的重建皮肤模型，10ug/mL和100ug/mL侧柏提取物可以有效的增加重建皮肤模型表皮的厚度，基底层上细胞排列更加紧密，角质层分化更加完善。这说明侧柏提取物可以提升皮肤整体活力和健康状况，具有修复皮肤组织的能力。
基于上述试验结果，本发明提供了侧柏在制备皮肤组织修复产品中应用，更具体地是提供侧柏提取物在制备皮肤组织修复产品中的应用。
由于侧柏具备修复皮肤组织的作用，其可以不断的促进皮肤细胞增殖和皮肤组织的再生，在一定程度上“抵消”各种现有皮肤细胞活力下降、数量减少导致的衰亡现象，这与通过抗氧化物质减少现有各种皮肤细胞受到的自由基和氧化应激损害有本质的不同，故侧柏同样可以应用在相关延缓皮肤衰老产品的制备中。
作为优选，所述侧柏为侧柏提取物；更优选地，所述侧柏提取物为侧柏醇提物；在本发明具体实施方式中，所述侧柏醇提物为乙醇提取物，更具体为70％的乙醇提取物；
具体的，所述侧柏乙醇提取物按照如下方法提取：
取侧柏枝叶，以10倍体积的70％乙醇超声提取，滤液旋转蒸发至干，得到侧柏乙醇提取物；
进一步地，所述侧柏乙醇提取物按照如下方法提取：
取侧柏枝叶10g，粉碎，于室温下以10倍体积的70％乙醇500W超声提取2次，每次30min，滤液合并，滤液旋转蒸发至干，得到侧柏乙醇提取物约1.3g。
作为优选，所述产品为化妆品、药品或保健品。
由以上技术方案可知，本发明发现中天然植物侧柏提取物能够显著地上调有助于皮肤细胞、组织修复的基因sirt1、nidi1和igf1，同时能够显著增加表皮的厚度，使基底层上细胞排列更加紧密，角质层分化更加完善，表明侧柏提取物可以提升皮肤整体活力和健康状况，具有修复皮肤组织的能力，进而可以不断的促进皮肤细胞增殖和皮肤组织的再生，延缓各种现有皮肤细胞活力下降、数量减少导致的衰亡现象。
附图说明
图1所示为10μg/mL侧柏提取物干预后3D皮肤模型切片快蓝染色结果；
图2所示为100μg/mL侧柏提取物干预后3D皮肤模型切片快蓝染色结果；
图3所示为10μg/mL侧柏提取物干预后nid1基因表达结果；
图4所示为10μg/mL侧柏提取物干预后igf1基因表达结果；
图5所示为10μg/mL侧柏提取物干预后sirt1基因表达结果；
图6所示为100μg/mL侧柏提取物干预后nid1基因表达结果。
具体实施方式
本发明公开了侧柏的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述应用已经通过实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述应用进行改动或适当变更与组合，来实现和应用本发明技术。
在本发明具体实施例中，涉及到对比试验，各试验组除了应有的区别外，其余试验环境和原材料等均保持一致。
以下就本发明所提供的侧柏的应用做进一步说明。
实施例1：侧柏提取物的生产
取侧柏枝叶10g，粉碎，于室温下以10倍体积的70％乙醇500W超声提取2次，每次30min，滤液合并，滤液旋转蒸发至干，得到侧柏提取物约1.3g。
实施例2：皮肤模型的制备及测试
(1)制备：从皮肤组织中分别提取人原代成纤维细胞(NHDF)及人原代角质细胞(NHEK)。将人原代成纤维细胞复苏，培养消化后与胶原混合进行真皮阶段培养3～4天，然后将使用饲养层培养的人原代角质细胞，消化接种到真皮阶段的皮肤模型表面，依次经过表皮浸没培养及气液相培养阶段，培养至18天得到可用于检测的3D皮肤模型。
(2)测试：用上述提取的侧柏提取物蒸干粉以培养基为溶剂配置终浓度为10mg/mL的侧柏提取物溶液，在皮肤模型培养至3天按照测试浓度加入提取物溶液，并以未加提取物溶液的模型为对照(NT)，18天后提取测试组和对照组皮肤模型中RNA进行检测，同时进行切片快蓝染色；
(3)RNA的提取
剪取组织约2*3mm，放入2mL EP管中；加入1mL Trizol，加入2颗钢珠，用组织破碎仪匀浆，50Hz，5min；9000rpm，4℃1min，将上清液转移到新的1.5mL EP管中；加入200ul氯仿，轻轻颠倒数次混匀，室温放置5分钟；9000rpm，4℃15min；转上层水相(约400ul)于新1.5mlEP管中，加入等体积的异丙醇，混匀，室温静置10min；9000rpm10min 4℃；弃上清，沉淀用预冷的70％无水乙醇洗1次，8000rpm5min 4℃；弃上清，空气干燥5-10分钟，溶于30ulDEPC水中；分光光度计测定RNA浓度。
(4)基因表达分析
根据QuantiGene R 2.0Plex Assay试剂盒进行RNA的纯化和基因定量分析，使用luminex平台对基因表达进行分析。
(5)结果
从图1和图2(镜检倍数相同)中可以看到，在添加侧柏提取物后，对比未处理组的重建皮肤模型，10ug/mL和100ug/mL侧柏提取物可以有效的增加重建皮肤模型表皮的厚度，基底层上细胞排列更加紧密，角质层分化更加完善。这说明侧柏提取物可提升皮肤整体活力和健康状况，具有修复皮肤组织的作用；
添加侧柏提取物(浓度为10μg/mL)后，结果如图3所示，与对照组(NT)相比，在皮肤模型中，基因nid1上调1.74倍；添加侧柏提取物(浓度为10μg/mL)后，结果分别如图4、5所示，与对照组(NT)相比，基因igf1表达上调1.74倍，基因sirt1表达上调1.53倍；添加侧柏提取物(浓度为100μg/mL)，如图6所示，与对照组(NT)相比，在皮肤模型中，基因nid1表达上调1.35倍，两个浓度下的侧柏提取物可以诱导有助于皮肤组织修复的基因的高表达，提高皮肤细胞和组织整体修复能力。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.侧柏在制备皮肤组织修复产品中应用。
2.根据权利要求1所述应用，其特征在于，所述侧柏为侧柏提取物。
3.根据权利要求2所述应用，其特征在于，所述侧柏提取物为侧柏醇提物。
4.根据权利要求1所述产品，其特征在于，所述产品为化妆品、药品或保健品。
5.侧柏在制备延缓皮肤衰老产品中应用。
6.根据权利要求5所述应用，其特征在于，所述侧柏为侧柏提取物。
7.根据权利要求6所述应用，其特征在于，所述侧柏提取物为侧柏醇提物。
8.根据权利要求5所述产品，其特征在于，所述产品为化妆品、药品或保健品。
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